CLEC4G Antibody

What is CLEC4G and why is it important in research applications?

CLEC4G (C-type lectin domain family 4 member G), also known as LSECtin, is a type II transmembrane glycoprotein approximately 40 kDa in size and 293 amino acids in length. It belongs to the C-type (Ca²⁺-dependent) lectin superfamily and contains:

• A short N-terminal cytoplasmic tail (amino acids 1-31)

• A 21 amino acid transmembrane region

• An extracellular region with:

  • Two N-linked glycosylation sites (amino acids 73 and 159)

  • A coil-coil neck domain (amino acids 96-136)

  • A C-type lectin-like domain (CTLD) (amino acids 165-289)

  • A C-terminal Ca²⁺-dependent carbohydrate-recognition domain (C-type CRD)

CLEC4G is significant in research due to its roles in:

• Glycan binding (mannose, GlcNAc, and fucose) in a Ca²⁺-dependent manner

• Viral recognition and binding (SARS coronavirus, Ebola virus, Japanese encephalitis virus)

• Potential neuroprotection against Alzheimer's disease progression through inhibition of Aβ generation

Which tissue types express CLEC4G and how can this influence antibody selection?

CLEC4G expression has been identified in multiple tissues:

When selecting antibodies, consider:

• For liver studies: Antibodies targeting membrane epitopes may be more effective

• For neuronal studies: Antibodies recognizing monomeric forms and cytoplasmic epitopes are preferable

• For experimental validation: Use appropriate positive control tissues (liver for high expression, neuronal tissues for cytoplasmic localization)

What are the optimal protocols for detecting CLEC4G in different experimental applications?

Flow Cytometry:

• Harvest cells (dendritic cells, transfected cell lines)

• Wash with PBS containing 0.5% BSA

• Block Fc receptors if working with immune cells

• Stain with anti-CLEC4G antibody (e.g., Mouse Anti-Human LSECtin/CLEC4G Monoclonal Antibody, MAB2947 at manufacturer's recommended dilution)

• Incubate for 30 minutes at 4°C

• Wash twice

• If using unconjugated primary antibody, stain with appropriate secondary (e.g., Allophycocyanin-conjugated Anti-Mouse IgG)

• Analyze using appropriate isotype controls

Immunohistochemistry:

• Fix tissue sections (formalin-fixed paraffin-embedded or frozen)

• For FFPE: Perform antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

• Block endogenous peroxidase and non-specific binding

• Apply anti-CLEC4G antibody at recommended dilution (e.g., 1:500-1:2000 for polyclonal antibodies like 18173-1-AP)

• Incubate overnight at 4°C

• Apply appropriate detection system

• Counterstain, dehydrate, and mount

Western Blotting:

• Prepare protein lysates from tissues or transfected cells

• Separate proteins by SDS-PAGE

• Transfer to membrane

• Block and incubate with anti-CLEC4G antibody (recommended dilution: 1:500-1:5000)

• Expected band size: approximately 40 kDa

• Note: Expression levels may require enrichment or use of transfected cells as positive controls

How can I validate the specificity of a CLEC4G antibody in my experimental system?

A robust validation strategy should include:

• Positive and negative controls:

  • Positive: Liver tissue, CLEC4G-transfected HEK293 cells

  • Negative: Non-transfected HEK293 cells, irrelevant transfectants

• Multiple detection methods:

  • Compare results across Western blot, IHC, and flow cytometry

  • Expected pattern: Membrane staining in liver, cytoplasmic pattern in neurons

• Blockade experiments:

  • Pre-incubate antibody with recombinant CLEC4G protein before staining

  • Signal should be significantly reduced or eliminated

• siRNA knockdown:

  • Reduce CLEC4G expression in appropriate cell types

  • Compare antibody staining in knockdown versus control cells

• Isotype controls:

  • Use matched isotype control antibodies at the same concentration

  • Example: For MAB2947, use MAB003 as control

What are the common pitfalls when using CLEC4G antibodies and how can they be avoided?

How can CLEC4G antibodies be used to investigate the role of this protein in Alzheimer's disease?

Recent research has identified CLEC4G as having potential neuroprotective functions in Alzheimer's disease (AD). Antibody-based approaches to study this relationship include:

• Expression analysis in AD progression:

  • Compare CLEC4G levels in AD vs. control brain tissues using validated antibodies

  • Key finding: AD patients show significantly lower CLEC4G expression compared to non-demented individuals

  • Methodology: Use anti-CLEC4G antibodies for immunohistochemistry on age-matched brain sections

• Co-localization studies with AD markers:

  • Double-label immunofluorescence with CLEC4G and BACE1 antibodies

  • Design: Use confocal microscopy with spectrally distinct fluorophores

  • Controls: Include single antibody staining to control for bleed-through

• Mechanistic investigation of CLEC4G-BACE1 interaction:

  • Co-immunoprecipitation with CLEC4G antibodies to pull down protein complexes

  • Western blot analysis to detect BACE1 in the precipitates

  • Validate with reciprocal co-IP using BACE1 antibodies

• Functional analysis in neuronal models:

  • Overexpress CLEC4G in neuronal cells and measure Aβ levels

  • Key finding: CLEC4G overexpression decreases Aβ40 in the culture media of iPSC-derived cortical neurons

  • Controls: Compare with vector-only transfected cells

• Age-dependent analysis in APP/PS1 mouse models:

  • Documented finding: CLEC4G expression decreases with age in APP/PS1 transgenic mice

  • Methodology: Compare immunostaining patterns between 6-month and 13-month-old mice

  • Quantification: Use image analysis software to measure staining intensity

What approaches can resolve contradictory findings when using different CLEC4G antibody clones?

When faced with conflicting results from different antibody clones:

• Epitope mapping analysis:

  • Determine the binding sites of each antibody clone

  • Different epitopes may be differentially accessible depending on protein conformation or processing

  • Method: Use peptide arrays covering the CLEC4G sequence

• Multiple detection methodologies:

  • Compare results across Western blot, immunoprecipitation, flow cytometry, and imaging

  • Discrepancies may be method-specific rather than antibody-specific

• Splice variant analysis:

  • CLEC4G has been observed to have multiple splice variants in brain tissues

  • Different antibodies may preferentially recognize specific variants

  • Solution: Use RT-PCR to identify which variants are present in your samples

• Post-translational modification considerations:

  • CLEC4G contains N-glycosylation sites that may affect antibody binding

  • Test with deglycosylated samples to determine if glycosylation impacts recognition

• Independent validation strategies:

  • Use genetic approaches (CRISPR/Cas9, siRNA) to modulate CLEC4G expression

  • Employ orthogonal methods such as RNA-seq to correlate with protein detection

  • Consider mass spectrometry-based validation

How can CLEC4G antibodies be utilized to study viral interactions and potential therapeutic applications?

CLEC4G functions as a receptor for several viruses, making it valuable for infectious disease research:

• Viral binding studies:

  • Use CLEC4G antibodies to block viral attachment to cells

  • Methodology: Pre-incubate cells with blocking anti-CLEC4G antibodies before viral challenge

  • Readout: Measure infection rates in blocked versus unblocked cells

• Mechanistic analysis of CLEC4G-viral interactions:

  • Immunoprecipitate CLEC4G with antibodies after viral exposure

  • Western blot for viral proteins in the precipitates

  • Mass spectrometry analysis of co-precipitated proteins

• Identification of binding domains:

  • Use domain-specific CLEC4G antibodies to determine which regions are critical for viral binding

  • Compare antibodies targeting the CTLD versus other domains

  • Correlate binding inhibition with viral entry inhibition

• Therapeutic antibody development:

  • Screen for antibodies that specifically block viral binding without disrupting normal CLEC4G function

  • Test antibody fragments (Fab, scFv) for improved tissue penetration

  • Evaluate humanized versions of effective blocking antibodies

• Comparative analysis across viral families:

  • CLEC4G binds to SARS coronavirus, Ebola virus, and Japanese encephalitis virus

  • Use antibodies to compare binding mechanisms across different viral families

  • Identify conserved versus virus-specific interaction patterns

Why might CLEC4G antibody staining patterns differ between cell types and disease states?

Several factors can explain variable staining patterns:

How can I optimize dual labeling protocols to simultaneously detect CLEC4G and its interaction partners?

For co-localization studies with CLEC4G and interaction partners (e.g., BACE1):

• Antibody compatibility:

  • Select primary antibodies from different host species (e.g., mouse anti-CLEC4G and rabbit anti-BACE1)

  • If same-species antibodies must be used, consider directly conjugated antibodies or sequential staining protocols

• Signal optimization:

  • For weak CLEC4G signals in neuronal tissues, use tyramide signal amplification

  • Balance signal strengths by adjusting antibody concentrations individually before combining

• Preventing cross-reactivity:

  • Include additional blocking steps between primary antibodies if using sequential protocols

  • Validate each antibody independently before combining

  • Include controls with each primary alone to check for cross-reactivity of secondaries

• Proximity ligation assay (PLA):

  • For detecting protein-protein interactions within 40nm

  • Use species-specific PLA probes against CLEC4G and BACE1 antibodies

  • Quantify PLA signals in different brain regions or cell types

• Confocal optimization:

  • Use spectral unmixing for fluorophores with overlapping emission spectra

  • Employ sequential scanning to prevent bleed-through

  • Include single-labeled controls on the same slide

What are the critical considerations when using CLEC4G antibodies in quantitative studies of disease progression?

For quantitative analysis of CLEC4G in disease contexts:

• Standardization protocols:

  • Use recombinant CLEC4G protein standards for calibration

  • Include the same positive control tissue in each experiment

  • Apply batch correction methods for samples processed at different times

• Quantification methods:

  • For IHC/IF: Use digital pathology software with validated algorithms for intensity measurement

  • For flow cytometry: Include calibration beads to normalize mean fluorescence intensity

  • For Western blot: Use housekeeping proteins appropriate for the tissue/condition

• Age and disease stage considerations:

  • CLEC4G expression decreases with age in APP/PS1 mice

  • Stratify analyses by age and disease severity

  • Correlate CLEC4G levels with established biomarkers of disease progression

• Statistical approaches:

  • Use appropriate statistical tests based on data distribution

  • Account for potential confounding variables (age, sex, postmortem interval)

  • Calculate minimum sample sizes needed for desired statistical power

• Validation across methodologies:

  • Confirm protein-level changes with transcript-level measurements

  • Consider single-cell approaches to account for cellular heterogeneity

  • Use orthogonal methods (e.g., ELISA and Western blotting) to validate quantitative changes

How can single-cell approaches with CLEC4G antibodies provide new insights into cell-type specific functions?

Single-cell analysis using CLEC4G antibodies can reveal:

• Cell-type heterogeneity:

  • Single-cell RNA-seq data shows CLEC4G is predominantly expressed in neurons (both excitatory and inhibitory)

  • Flow cytometry with CLEC4G antibodies can identify and isolate CLEC4G-positive subpopulations

  • Combine with other neural markers to characterize CLEC4G-expressing neuronal subtypes

• Developmental regulation:

  • Higher CLEC4G expression in neural progenitor cells compared to differentiated neurons

  • Use antibodies to track expression changes during neural differentiation

  • Correlate with functional maturation markers

• Disease-specific changes:

  • Percentages of CLEC4G-positive cells differ between AD (1.44%) and control (1.80%) neuronal populations

  • Expression levels within positive cells are higher in controls versus AD

  • Use CyTOF or spectral flow cytometry to create comprehensive profiles of CLEC4G-expressing cells

• Spatial context:

  • Combine with spatial transcriptomics or multiplexed imaging

  • Map CLEC4G expression patterns in relation to brain regions and pathological features

  • Identify microenvironmental factors influencing expression

What role might CLEC4G play in glycosylation-related pathways relevant to neurodegeneration?

CLEC4G's role in glycan binding suggests connections to neurodegeneration:

• N-glycan interactions:

  • CLEC4G has high affinity for GlcNAc (N-acetylglucosamine)

  • N-glycan structures are crucial in AD progression

  • Research approach: Use glycosidase treatments to modify N-glycans and assess impact on CLEC4G binding/function

• Correlation with glycosylation enzymes:

  • Significant negative correlations observed between CLEC4G and:

    • MGAT1 (AD: r = -0.45, p < 0.0001; Control: r = -0.30, p < 0.0001)

    • MGAT3 (AD: r = -0.17, p < 0.05; Control: r = -0.22, p < 0.0001)

  • Experimental approach: Co-immunoprecipitation of CLEC4G with glycosylation enzymes

• Functional implications:

  • CLEC4G may limit N-acetylglucosamine accumulation in neurons

  • N-acetylglucosamine on BACE1 is elevated in AD patients

  • Investigation strategy: Measure N-acetylglucosamine levels in CLEC4G-overexpressing versus control neurons

• Therapeutic potential:

  • CLEC4G's ability to recognize specific glycan structures could be leveraged for targeted interventions

  • Approach: Screen for compounds that enhance CLEC4G-mediated clearance of pathological glycans

What are the latest advances in using CLEC4G antibodies for understanding transcriptional regulation in neuronal systems?

Recent research has identified key transcriptional regulators of CLEC4G:

• Upstream transcription factors:

  • NR2F6 and XRCC4 have been identified as primary regulators of CLEC4G in neuronal cells

  • Research methodology: Use chromatin immunoprecipitation (ChIP) with antibodies against these factors to confirm binding to CLEC4G promoter

• Regulatory network differences:

  • Transcription factor modules differ between high-CLEC4G (control) and low-CLEC4G (AD) neuronal cells

  • Approach: Use SCENIC (Single-Cell Regulatory Network Inference and Clustering) with antibody-based cell sorting

• Modulation strategies:

  • Target NR2F6 and XRCC4 to potentially upregulate CLEC4G expression

  • Experimental design: Overexpress these factors in neuronal cells and measure CLEC4G levels

  • Validation: Use CLEC4G antibodies to quantify protein changes following transcription factor modulation

• Epigenetic considerations:

  • Investigate methylation status of CLEC4G promoter in different brain regions

  • Combine with antibody-based detection to correlate epigenetic marks with protein expression

  • Approach: Use methylation-specific antibodies alongside CLEC4G antibodies